DNA extraction is a sample preparation technique often utilized in clinical and forensic applications to purify and concentrate DNA for genetic analysis from small volume samples that typically are dilute or biologically complex, e.g. blood. Significant effort has been invested in the last two decades into devising methods that reduce the amount of sample required for genetic analysis, often to address the needs of clinical and forensic communities. One such method has involved adapting traditional genetic analysis methodologies to a microscale format. The miniaturization of sample preparation techniques, including DNA extraction, has been included in the move towards microscale analysis. Such miniaturization has been found to minimize sample handling and contamination, as well as helping to reduce analysis time. Solid phase extraction (SPE), the current DNA sample preparation technique of choice in clinical and forensic laboratories, is among the techniques that have been miniaturized for microscale analysis. Micro-SPE (μSPE) columns have been developed in both capillaries and microdevices such as microfluidic chips (Tian et al., Anal. Biochem. 2000, 283, 175-191; Wolfe et. al. Electrophoresis 2002, 23, 727-733; Breadmore et al. Anal. Chem, 2003, 75, 1880-1886).
In the presence of a chaotropic solution, nucleic acids bind avidly to a hydrophilic silica surface. This has been described previously and represents the chemical basis for the most common form of SPE for DNA samples. The most widely used silica-based SPE column for DNA extraction is fabricated using silica-based particles or beads (Melzak et al. J. Colloid Interf. Sci. 1996, 181, 635-644). DNA extraction and purification has been achieved with good efficiency in microscale formats utilizing silica beads in μSPE columns. Issues of reproducibility, however, have resulted from the inability to completely immobilize the silica beads within the column (Wolfe et al. Electrophoresis 2002, 23, 727-733). This problem has been addressed using the dual weir-type approach described in Anal. Chem. 2000, 72, 585-590 and a bead immobilization method described in Anal. Chem. 2003, 75, 1880-1886. In the latter method, silica beads were packed into the microchannels of a microfluidic glass chip and immobilized with a “nano-glue” comprised of a tetraethoxyorthosilicate (TEOS) based sol-gel. This provided a continuous and stable solid phase μSPE column for DNA extraction.
Despite improvements made to silica bead based μSPE columns, fabrication of this type of solid phase column within microdevices, particularly through bead packing, has several distinct disadvantages. First, the additional processes involved in filling microchannels on devices such as microfluidic chips with silica beads increases the fabrication time for such solid phase columns. Second, when using microfluidic chips for extraction, chip-to-chip extraction reproducibility, while somewhat improved through bead immobilization, continues to be a significant problem. Third, while the surface area available for DNA binding is enhanced by decreasing the bead diameter, smaller diameter beads (e.g., 5 μm) are more difficult to contain, resulting in higher back pressures which limit μSPE columns in microdevices to relatively low flow rates and low binding capacity. Several prior art examples have demonstrated that bead packing problems can be eliminated by providing a high surface area-to-volume ratio in a μSPE chamber through the etching of pillars in the chamber during fabrication (Cady, N. C.; Stelick, S.; Batt, C. A. Biosens. Bioelectron. 2003, 19, 59-66; Christel, L. A.; Petersen, K.; McMillan, W.; Northrup, M. A. J. Biomed. Eng. 1999, 121, 22-27). While this increases the surface area for DNA binding and provides a regular array for reproducible chromatography, complex fabrication requirements and cost make these microdevices less attractive. Moreover, a large volume of elution buffer (greater than 50 μL) is required to elute the bound DNA from the columns of these microdevices, creating potential difficulties with downstream processing (e.g., PCR).
The fabrication of silica- and organic polymer-based rigid, porous monolithic columns has been reported as an alternative to using silica beads in HPLC and SPE applications and has provided new possibilities for the fabrication of μSPE columns in microdevices. Such monolithic columns are fabricated in situ by thermal- or photo-induced polymerization of a solution of monomer, initiator, and porogenic solvent. The resulting solid phase comprises pores in the nanometer to micron size range with a continuous interconnected network of channels. The advantages of in situ polymerization, including pore size control, high flow-rate and large mass-transfer, have allowed them to be successfully used in capillary electrochromatography and pre-concentration applications, e.g., chemical compounds, peptides and proteins.
Thermally-induced polymerized monolithic columns have been demonstrated as a functional medium for DNA separations by HPLC on commercial flat-disk CIM® (BIA Separations) monolith columns. DNA purification and separation have also been performed on bacterial and yeast genomic DNA in these columns; however, these columns showed low extraction efficiencies and required a high salt and high pH buffer for DNA release, which has been found to interfere with downstream processing, e.g. PCR. Moreover, thermally-induced polymerization does not ensure the accurate placement of monolithic columns within the architecture of microdevices. By contrast, UV initiated photo-polymerized monolithic columns can be formed within specified spaces. As a result, both silica- and organic polymer-based photo-polymerized monolithic columns have been incorporated into microdevices (Morishima et al. J. Anal. Chem. 2001, 73, 5088-5096).
While silica-based monolithic columns have been found to be functional for binding and extracting DNA, the use of certain silica-based monomers as part of the initial polymerization mixture has been problematic. Tetraethylorthosilicate (TEOS)-based monolithic columns have been found not to yield extraction efficiencies comparable to columns created with silica beads, due mainly to the difficulty in controlling pore size within such silica-based sol gel columns which, in turn, inhibits fluid flow. Silica-based monolithic columns reported by Ferrance et al. Anal. Chim. Acta 2003, 500, 223-236, were produced using tetramethoxyorthosilicate (TMOS) monomers and a porogen to provide the appropriate pore size, but these columns could not easily be localized within microdevices.
None of the methods described above provides the important advantages of the fabrication method for a grafted UV photo-polymerized silica-based monolithic column. These advantages include increased column capacity and efficiency for DNA extraction resulting in significantly higher DNA yields from very low volume DNA samples of the type encountered in clinical and forensic applications; precise placement of the monolithic column in a capillary or other microdevice, such as a microfluidic microchip; minimal reagent volume required to elute DNA from the column; and the ability to use a low ionic strength buffer for the elution of DNA from the monolithic column, allowing for direct PCR analysis of the extracted DNA without further sample cleaning steps.